Cloning and molecular characterization of△^(12)-fatty acid desaturase gene from Mortierella isabellina

AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

Construction of a High-efficient Expression Vector of Δ^(12) Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

A full-length sequence coding for △^12 fatty acid desaturase gene from peanut（Arachis hypogaea L.）was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21（DE3）pLysS. The △^12 fatty acid desaturase was highly expressed in E. coli BL21（DE3）pLysS in the presence of isopropyl-D-thiogalactopyranoside （IPTG）. The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20℃ for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS （gas chromatogram and gas chromatogram-mass spectrometry） analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited △^12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.

Comparison of the Δ^(12) fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes

△^12 fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds. By RT-PCR method, the full-length cDNAs of △^12 fatty acid desaturase gene were isolated from peanut （Arachis hypogaea L.） genotypes with normal and high ratio of oleic to linoleic acid, which were designated AhFAD2B and AhFAD2B＇, respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position ＋442 bp of AhFAD2B＇ sequence of high oleic acid genotypes, which resulted in the shift of open reading frame and a truncated protein AhFAD2B＇, with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of △^12 fatty acid desaturase gene in high oleic acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast （Saccharomyces cerevisiae） cell transformed with AhFAD2B or AhFAD2B＇ proved that only AhFAD2B gene product showed significant △^12 fatty acid desaturase activity, but AhFAD2B＇ gene product did not. These results suggested that the change of AhFAD2B＇ gene sequence resulted in lower activity or deactivation of △^12 fatty acid desaturase in high oleic acid genotype.